GSM6810450: ScRNA-seq, mRNA library; Mus musculus; RNA-Seq
Single cell suspensions of spleens were obtained by pressing organs through a 70-μm nylon mesh cell strainer with a plastic plunge in PBS 5%BSA 1mM EDTA. Splenic cell suspensions were incubated with red blood cell lysis buffer for 3 minutes at room temperature and enriched for CD4+ T cells by depletion of CD8+ and CD19+ cells using the autoMACS® Pro Separator with the Deplete program (Miltenyi). We used cell hashing with hashtag oligonucleotides (HTO) to multiplex the two samples from 2 individual mice. Cell surface staining used for gating Foxp3+ Treg cells and staining for distinct barcoded anti-mouse CD45 antibody (Biolegend; A0308 and A0307 for WT and Lta-/- Treg cells, respectively) were performed in PBS 5%BSA 1mM EDTA for 30 min on ice. For each sample, Foxp3+ Treg cells (Live Dead-CD4+Foxp3eGFP+) were bulk-sorted with BD FACS Aria II. Sorted cell samples (20,000 WT Treg cells and 20,000 Lta-/-Treg cells) were pooled and loaded in a single capture well for subsequent 10x Genomics Single Cell 3' v3.1 workflow. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, Foxp3+ Treg cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The resulting libraries were pooled and sequenced together on an Illumina NextSeq2000 platform with P2 flow cells (100 cycles).